The characterization of biotherapeutics often involves assessing and comparing different approaches to analyze and measure protein degradations and modifications, such as deamidation, oxidation, and glycosylation. These modifications are deemed critical quality attributes (CQAs) due to their ability to affect drug efficacy. To gain a thorough understanding of these and other protein modifications, mass spectrometry (MS) analyses are frequently conducted on both intact proteins and peptide fragments (bottom-up approach). However, there are a lack of methods available to directly compare and correlate the results of these orthogonal approaches.
The Intact Reconstruction workflow is a multi-node workflow that allows the user to correlate deconvoluted intact data with peptide mapping data. Modifications at the residue level are quantified using the PTM workflow and these values are then convolved against each other to reconstruct an intact mass spectrum. This reconstructed spectrum is then compared with the experimental data for evaluation.
This method can be used to quickly evaluate the alignment of orthogonal methods for PTM quantification, degredation or conjugation analyses. It also provides a useful way to determine if acquisition parameters require optimization.
Method
The first step in Project Creation is to load in the intact and peptide map raw data files in the Samples tab. It's important make sure there's a string of unique text in each raw file. This will be used to point to the appropriate node during the processing stage. By default, this text is Intact and Peptide.
In the Sequences and Masses tab, load the FASTA files:
Three entries should be created under Sequence combinations: One single LC, one single HC,
and one entry containing two of each chain.
In the Processing Nodes tab, make sure that the same unique text that appears in the sample name is used to point to the appropriate processing node:
Enter the processing parameters, including a full list of modifications in the Byonic node and click Create Project.
On completion, two project files will be created (.blgc and .ntms) along with a PTM report.
The PTM report contains a tab named Reconstruction Export. This table should be exported as shown below:
( Note: residue positions should not be more than 100% modified)
Open the intact project (Inspection view) and select the Reconstruct mass option in the Deconvoluted Mass window.
From the Reconstruction box, select Add CSV import and browse to the file created from the PTM report.
In the Protein Targets box use the Add row button to add rows for the HC and LC and adjust the protein count accordingly.
Gauss width sets the width of the reconstruction peak and will depend on the instrument resolution (values between 5 and 20 are good starting points).
If the instrument calibration is off, then the Mass Offset can be used to correct for this.
Select Ok and the reconstruction will take place. The experimental Intact deconvolution is shown in black, and the theoretical reconstruction in blue: